Dynamic adsorption/desorption of proteins with thermo-responsive polymer grafted sepharose fast flow sorbents

Abstract

This investigation has focused on the dynamic adsorption and desorption behaviour of proteins with thermo-responsive co-polymer grafted chromatographic materials. To that end, the dynamic adsorption performance of bovine holo-lactoferrin (bhLf) with two thermo-responsive chromatographic sorbents, i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-acrylic acid) grafted Sepharose Fast Flow (ITA) and poly (NIPAAm-co-tert-butylacryl amide-co-2-(diethylamino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD) at two different column temperatures and different protein loading conditions was examined by frontal chromatographic analysis methods. The dynamic binding capacity (DBC), dynamic binding rate (DBR) and column efficiency of the ITA sorbent for bhLf at 50 °C were greater than those at 20 °C. The dynamic binding characteristics were observed to be influenced by the protein feed concentrations, showing an increase of binding capacity with an increase in the bhLf feed concentration. A large proportion (≥65%) of the bhLf molecules adsorbed onto the ITA sorbent could be eluted from the column by simply lowering the column temperature from 50 °C to 20 °C. The corresponding DBC values of ITA sorbent for bovine serum albumin (BSA) were much lower than those for bhLF at both 20 °C and 50 °C due to electrostatic repulsion effects between the negatively charged BSA and the negatively charged co-polymer modified surface of the ITA sorbent, whilst the opposite trend was found with positively charged co-polymer (i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-2-(diethyl-amino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD), indicative of different separation selectivity.